Characterization of putrescine transport across the intestinal epithelium: study using isolated brush border and basolateral membrane vesicles of the enterocyte

Abstract. Putrescine transport was investigated in isolated brush border and basolateral membrane vesicles prepared from the rabbit enterocyte. Brush border vesicles were oriented right-side-out and basolateral vesicles inside-out, forming a model representing uptake and extrusion across the intestinal epithelium. Putrescine transport across both membranes was initially rapid, and 66% of the equilibrium uptake was achieved within the first minute. According to osmo-plots and measurements at 4C, 20% of total incorporation presented binding to the membrane. In order to estimate actual uptake into the vesicles, K_m was calculated from the differences in putrescine incorporation at 37C and 4C, and was 12.7 μmol L^-1 for brush border uptake and 38.2 μmol L^-1 for basolateral extrusion. Putrescine uptake into brush border and basolateral membrane vesicles was not enhanced in the presence of an Na⁺ gradient. When Na⁺ was substituted with an uncharged solute, mannitol, putrescine incorporation was increased, indicating that putrescine uptake is not Na⁺-dependent and that cations might interfere with the carrier. Paraquat and methylglyoxal-bis(guanylhydrazone), known to share the polyamine transport system, inhibited putrescine incorporation in both membrane vesicle preparations. Basolateral carrier showed significantly higher sensitivity to cations. We conclude that putrescine uptake across the apical membrane and extrusion across the basolateral membrane of the enterocyte are mediated by two different and independent carriers which differ in their electrical properties.     

Insulin induced hypoglycaemia and sugar transport across the brush border and basolateral membranes of rat jejunal enterocytes

Abstract. Acute hypoglycaemia enhances intestinal sugar uptake but the mechanisms involved are unknown. Results from the present study show increased galactose movement across the brush border and basolateral membranes of isolated upper, but not mid-villus, jejunal enterocytes 45 min after intravenous administration of insulin to rats at a level which reduced by half plasma glucose concentration. Incubation of upper villus cells from uninjected animals with insulin (100 mU ml^-1) for 40 min was without effect on brush border or basolateral sugar transport. Insulin treatment of rats did not affect glucose uptake by brush border vesicles prepared from upper villus cells when the process was driven by an inwardly directed 100 mM sodium thiocyanate gradient. In contrast, glucose uptake using a 100 mM inwardly directed sodium chloride gradient was reduced by 49% following hypoglycaemia. It is concluded that the enhanced sugar uptake following insulin hypoglycaemia involves both brush border and basolateral membranes of only the most mature villus cells at the villus tip. Upregulation of Na⁺-sugar cotransport at the brush border is best explained by an increased electrochemical driving force for Na ⁺-sugar cotransport rather than increased numbers of transporters. The transport response is not due to a direct effect of insulin on the enterocyte and the possible systemic factors involved are discussed.     

The subcellular localization of peptidase activity in the human jejunum

Abstract. The subcellular localization of peptidase activity in the normal human jejunum has been investigated. Subcellular organelles were fractionated by density gradient centrifugation. The localization of peptidases was determined by comparing the distributions of peptidase activities with those of organelle 'marker' enzymes. The organelles and their markers were: cytosol—lactate dehydrogenase; brush border—neutral α-glucosidase, γ-glutamyl transferase and leucyl-2-naphthylamidase; plasma membrane—5'-nucleotidase; lysosomes—N-acetyl- β -glucosaminidase; mitochrondria—malate dehydrogenase; endoplasmic reticulum—alkaline α-glucosidase; peroxisomes—catalase.
Thirteen dipeptides, seven tripeptides, two tetrapeptides, two pentapeptides and a hexapeptide were used as substrates. The distribution of dipeptidyl peptidase IV was also determined.
Irrespective of whether the NH₂-terminal or COOH-terminal amino acid was neutral, basic or acidic, the major or exclusive locus of dipeptidase activity was cytosolic. All of the activity against a dipeptide with the amino acid proline at the NH₂-terminus was in the cytosol.
The distribution of tripeptidase activity was quite different. Although the cytosol hydrolysed all tripeptides, as much as 50% of tripeptidase activity was particulate. For both tetrapeptides, one of the pentapeptides and the hexapeptide, the major or exclusive locus of activity was the brush border membrane. Pentaphenylalanine, however, was hydrolysed by both the cytosol and the brush border. Dipeptidyl peptidase IV was localized in the brush border.     

Systemic lupus erythematosus is associated with increased auto-antibody titers against calreticulin and Grp94, but calreticulin is not the Ro/SS-A antigen

Abstract Auto-antibodies against purified human calreticulin were determined by an ELISA in sera from patients with systemic lupus erythematosus (SLE) and from healthy persons or patients without an autoimmune disease. More than 80% of patients with SLE had titers exceeding the highest value obtained in the group without SLE. Almost 30% of the patients had also elevated auto-antibody titers against purified rat grp94, another resident ER-protein of the KDEL-protein family, but not against rat ERp72 (CaBP2), an ER-resident protein of the proteindisulfide isomerase family. It could, however, be excluded that calreticulin is the Ro/SS-A antigen on the basis of the following observations: 1) Calreticulin purified from rat, bovine or human liver contained far less than 1 mol of phosphate per mol of calreticulin, showed an E280/E260-absorption ratio of about 2·0, and did not contain extractable RNA; 2) Sera from patients with SLE did not react with or precipitate endogenous calreticulin from Hep G2 cells; they did, however, precipitate hY-RNA from these cells; 3) Sera from SLE-patients, but not anti-calreticulin antisera precipitated [³²P]-hY-RNA from [³²P]-labelled Hep G2 cells.     

Tritium and 14C isotope effects using tracers of leucine and alpha-ketoisocaproate

Abstract. To test if different leucine tracers behave in an indistinguishable manner and, by implication, that their metabolism is identical to that of natural leucine, we measured whole body leucine turnover in dogs and humans and fibrinogen synthesis in dogs by simultaneously infusing either [1–¹⁴C]leucine or [4,5–³H]leucine or [I-¹⁴C]α-ketoisocaproate (KIC) and [4,5–³H]KIC. Whole body leucine fluxes calculated from the plasma specific activity of the transaminated product of the infused tracer (reciprocal pool model) were lower (dogs by 5.7%; humans by 6.4%, both P<0.02) when the plasma 'H specific activity compared to ¹⁴C specific activity were used with leucine tracers and were also lower (dogs by 4.4%, P<0.02; humans by 86%, P<0.06 ) using the KIC tracers. Using leucine or KIC tracers in dogs, the fractional rate of fibrinogen synthesis was 6.7% or 9.4% lower, respectively, (P<0.02) using the ³H versus the ¹⁴C tracer. The apparently lower incorporation of ³H into protein was only in part accounted for by detritiation (2.1%, P = 0.05) of [³H]leucine during acid hydrolysis of proteins. These results suggest that in vivo and/or in vitro differential isotope effects are small (˜5%), but should be considered when dual isotopes infusions are employed to partition amino acid metabolism.     

Effect of netilmicin on the phospholipid composition of subcellular fractions of rat renal cortex

The purpose of this study was to determine the subcellular site(s) of the renal cortical phospholipidosis induced by aminoglycosides. For this purpose we injected male Sprague-Dawley rats s.c. with netilmicin, containing tracer quantities of [3H]netilmicin, at 100 mg/kg/day for 2 days; control rats were injected with saline. Twenty-four hours after the second injection of drug the rats were sacrificed and the renal cortex was fractionated by differential ultracentrifugation and Percoll gradient density techniques to obtain purified lysosomes, mitochondria, microsomes, brush border membranes and basolateral membranes. The total phospholipid content of the renal cortex was 300 +/- 5 nmol/mg of protein in control rats and 340 +/- 5 nmol/mg of protein in netilmicin-injected rats. The total phospholipid content of the lysosomal fraction of netilmicin rats, which was enriched in myeloid bodies and [3H]netilmicin, was 91% greater than that of control rats and reflected significant increases of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. This pattern is identical to that reported previously for the rat renal cortical phospholipidosis induced by aminoglycosides. The total phospholipid contents of the mitochondrial, microsomal, brush border membrane and basolateral membrane fractions of netilmicin-injected rats were higher by approximately 10% than the respective fractions of control rats and each fraction exhibited a significant increase of one or more of the four phospholipids elevated in the renal cortical homogenate and in the lysosomal fraction. The data indicate that the myeloid body is the primary source of the lysosomal phospholipidosis induced by netilmicin which provides support for the hypothesis that the lysosomal phospholipidosis is secondary to aminoglycoside-induced inhibition of phospholipid degradation. In addition the findings of increased phospholipid content and altered phospholipid composition of the other subcellular fractions raise the possibility that aminoglycoside antibiotics cause a more generalized disturbance of phospholipid metabolism characterized by altered synthesis as well as degradation in renal proximal tubular cells.     

Effect of activation of protein kinase A and of protein kinase C on the kinetics of the renal basolateral PAH transporter

Summary— The aim of the present study was to examine the effect of activation of the protein kinase A (PKA) and protein kinase C (PKC) pathways on ³H-p-aminohippurate (PAH) uptake of isolated S₂ segments of proximal tubules, microdissected from rabbit kidneys without the use of enzymatic agents. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of ³H-PAH reflects transport across the basolateral membrane. The phorbol ester phorbol 12-myristate 13-acetate (PMA) (10 ⁷ M), an activator of PKC, significantly increased tubular ³H-PAH uptake with steady state conditions (by 115%), whereas dibutyryl cyclic adenosine monophosphate (db-cAMP) (10^-4 M) and forskolin (10^-4 M) significantly inhibited it (by 42% and 52%, respectively). Kinetic data, which were based on 15 sec PAH uptake measurements, revealed that PMA, after a 10 min incubation period, significantly enhanced Km and Vmax of the PAH transporter (Km from 174 ± 22 to 447 ± 91 μM, Vmax from 2.76 ± 0.24 to 16.67 ± 1.85 pmol nL^-1 min^-1), whereas db-cAMP significantly decreased Vmax (from 2.76 ± 0.24 to 1.82 ± 0.19 pmol nL^-1 min^-1). The Km value was also numerically lowered by dibutyryl-cAMP (from 174 ± 22 to 139 ± 21 μM), but this change did not reach statistical significance. The data provide evidence that short time activation of the PKC pathway 1) enhances the effectiveness of PAH transport into proximal S₂ segments across the basolateral cell membrane, 2) increases the maximum transport rate of the PAH transporter and 3) decreases its affinity for PAH. Activation of the cAMP/PKA pathway induces the opposite effects.     

Membrane distribution of sodium-hydrogen and chloride-bicarbonate exchangers in crypt and villus cell membranes from rabbit ileum.

Present evidence suggests that in the small intestine, villus cells are primarily absorptive and crypt cells are primarily secretory. In order to further confirm that there are differences in transport properties between villus and crypt cells, we have separated villus from crypt cells, using calcium chelations techniques, and determined the distribution of Na:H and Cl:HCO3 exchange activity on brush border membrane and basolateral membrane preparations from these two cell populations. Separation of cells was determined utilizing alkaline phosphatase and maltase activity as a marker of villus cells and thymidine kinase activity as a marker of crypt cells. Utilizing these techniques, we were able to sequentially collect cells along the villus-crypt axis. Na-stimulated glucose and alanine uptake in brush border membrane vesicles diminished from the villus to the crypt region in the sequentially collected cells fractions, further suggesting separation of these cells. Brush border and basolateral membranes were then prepared from cells from the villus and crypt areas, utilizing a continuous sucrose gradient. In the villus cells, Na:H exchange activity was found associated with both the brush border and basolateral membrane, whereas, in crypt cells, Na:H exchange activity was only found on the basolateral membrane. Cl:HCO3 exchange activity was found only on the brush border membrane, in both villus and crypt cells. These studies suggest functional heterogeneity in ion transport between villus and crypt cells.     

Pharmaceutical preparation of oxygen-15 labelled molecular oxygen and carbon monoxide gasses in a hospital setting

Background:  Clinical positron emission tomography (PET) requires safe and effective PET radiopharmaceuticals. Tracers used for measuring oxygen consumption and blood volume are [¹⁵O]O₂ and [¹⁵O]CO, respectively. In general, these oxygen-15 labelled tracers are produced using a cyclotron that accelerates deuterons onto a target filled with ¹⁴N₂ containing a trace of oxygen. In recent years, cyclotrons have been developed that only are capable of accelerating protons. The purpose of this study was to validate and assess such a cyclotron for production and administration of oxygen-15 labelled gasses in an hospital setting.
Methods:  An RDS111 cyclotron (Siemens-CTI, Knoxville, USA) was validated for bolus production of [¹⁵O]O₂ and [¹⁵O]CO gasses. In addition, equipment was developed to administer these tracers to patients.
Results:  Both [¹⁵O]O₂ and [¹⁵O]CO gasses could be produced in sufficient amounts, whilst meeting European Pharmacopeia requirements. Although produced oxygen-15 gasses contained a minor level of ¹¹C contamination, in clinical studies it was possible to correct for this contamination by delayed blood counting.
Conclusion:  An 11 MeV proton cyclotron combined with an in-house developed gas delivery system allows for the production and administration of sufficient amounts of [¹⁵O]-gasses for routine clinical PET studies in an hospital setting.     

Effects of triiodothyronine and dexamethasone on plasma and tissue angiotensin converting enzyme in the rat

Summary— In order to identify tissue specific regulation of angiotensin converting enzyme (ACE), the effects of dexamethasone (0.04 mg sc per day for 7 days) and triiodothyronine (T3) (0.5 mg/kg sc per day for 10 days) on ACE activity were investigated in different tissues in male Wistar rats. ACE activity was measured by fluorimetry in the plasma, heart, lung and kidney. In the kidney, ACE activity was measured in the medulla, cortex and brush border of proximal tubular cells and ³H-ramiprilat binding was used to characterise the changes in brush border ACE activity. Dexamethasone elicited a significant increase in lung ACE activity and a significant decrease in plasma ACE activity, but did not alter enzyme activity in the other tissues studied. T3 produced a significant decrease in lung ACE activity and an increase in ACE activity in the plasma and heart. In the kidney, ACE activity was not modified in the medulla whereas in the cortex and brush border ACE activity was doubled. This increase in ACE activity corresponded to a similar increase in the maximum number of binding sites of ³H-ramiprilat, suggesting that the increase in activity corresponded to an increase in the ACE level. The increased heart and kidney ACE activity in response to T3 may contribute to the cardiovascular effects of thyroid hormones through increased local angiotensin II generation. These results show that under dexamethasone or T3, ACE activity can vary from one tissue to another, suggesting that the ACE regulatory mechanism acts differently in each tissue.     

Cytoplasmic regions of the β3 subunit of integrin αIIbβ3 involved in platelet adhesion on fibrinogen under flow conditions

Summary.  Platelet adhesion to surface-bound fibrinogen depends on integrin α_IIbβ₃. In the present study, we investigated the role of the regions ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T of the β₃ cytoplasmic tail in the regulation of platelet adhesion under flow conditions, by introducing peptide mimetics in platelets. Introduction of peptide EATSTFTN (E–N) increased surface coverage by 35%, an effect caused by 25% more adhesion. In contrast, peptide TNITYRGT (T–T) decreased surface coverage by 16%, as a result of 25% less adhesion. An S→P substitution in the E–N peptide, thereby mimicking a mutation in Glanzmann's thrombasthenia, abolished the effect of E–N. A suboptimal concentration of cytochalasin D is known to enhance ligand binding to α_IIbβ₃ in platelet suspensions. Under flow, cytochalasin D (1 µmol L⁻¹) induced 50% more platelet adhesion, with a strong reduction in platelet spreading. Both peptides opposed the increase in adhesion by cytochalasin D and partly (E–N) and completely (T–T) restored platelet spreading. Thus, the ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T regions of β₃ contribute to the regulation of αIIbβ3 anchorage to the cytoskeleton and platelet spreading to an adhesive surface.     

Specific interaction of food proteins with apical membranes of the human intestinal cell lines Caco-2 and T84

A comparison between the intestinal epithelial cell lines Caco-2 and T84 was made to assess the influence of enterocytic differentiation on food protein binding capacities of the brush border membrane. Cell morphology and expression of brush border-associated enzymes were studied as differentiation markers. Food protein binding to isolated brush border membranes was measured with a dot blot chemiluminescence assay. Early at confluence, Caco-2 cells exhibited a more differentiated state compared to T84 cells. Brush border membranes of both cell lines bound gliadin peptides, β-lactoglobulin and ovalbumin specifically. Binding capacities increased from gliadin peptides to ovalbumin to β-lactoglobulin. There was correlation of membrane binding capacity with degree of cell differentiation. Due to their similarity to small intestinal epithelial cells, the colon carcinoma cell lines Caco-2 and T84 represent models for studying food protein–enterocytic brush border membrane interactions in relation to varying degrees of cell differentiation.     

Mechanism of vancomycin transport in the kidney: studies in rabbit renal brush border and basolateral membrane vesicles.

The effect of vancomycin, a putatively nephrotoxic amine glycopeptide antibiotic, on the transport of organic cations was examined in rabbit renal basolateral and brush border membrane vesicles. The studies were conducted using a rapid filtration technique and the prototypic organic cation tetraethylammonium. In basolateral membrane vesicles, vancomycin cis-inhibited the electrogenic transport of tetraethylammonium with an IC50 value of 260 microM. In contrast, gentamicin, an aminoglycoside, was without effect. Inhibition by mepiperphenidol, a classical organic cation transport inhibitor, was observed with an IC50 value of 24 microM. Countertransport, that is, trans-stimulation experiments, were initiated in order to determine whether or not vancomycin was capable of traversing the plasma membrane. Vancomycin caused trans-stimulation of tetraethylammonium uptake. The specificity of inhibition was assessed by determining the effect of vancomycin on the transport of p-aminohippurate, an organic anion. Vancomycin did not inhibit transport, whereas probenecid, a classical organic anion inhibitor, did. In the brush border membrane, vancomycin had no effect on the transport of tetraethylammonium. These data are consistent with mediated transport for vancomycin across the basolateral membrane, but not across the brush border membrane. This implies that the nephrotoxicity of vancomycin may be due to entry through the basolateral membrane and the absence of mediated egress at the brush border membrane.     

The in vitro pharmacological profile of KR31080, a nonpeptide AT1 receptor antagonist

Summary— KR31080 (2-butyl-5-methyl-6-(1-oxopyridin-2-yl)-3-[[2'-(1H-tetrazol-5-yl) biphenyl-4-yl]methyl]-3H-imidazo[4,5-b] pyridine) is a potent inhibitor of angiotensin type 1 (AT₁) receptors in rabbit aorta and human recombinant AT₁ receptors. In the isolated rabbit thoracic aorta, KR31080 caused a nonparallel shift to the right of the concentration-response curves to angiotensin II (All) with decreased maximal response (pD'₂ = 10.1 ± 0.1), but had no effect on the contractile response induced by norepinephrine. KR31080 inhibited specific [¹²⁵I]AII binding to rabbit aortic membranes (AT, receptors) and [¹²⁵I][Sar¹, Ile⁸]AII binding to human recombinant AT₁ receptors in a concentration-dependent manner with IC₅₀ values of 0.84 ± 0.08 nM and 1.92 ± 0.15 nM, respectively, but did not inhibit specific [¹²⁵I)AII binding to bovine cerebellum membranes (ÀT₂ receptors). In the Scatchard analysis, KR31080 interacted with rabbit aortic AT₁ receptors in a competitive manner, similar to losartan. These results demonstrate that KR31080 is a potent and AT₁ selective angiotensin receptor antagonist which exerts a competitive antagonism in the [¹²⁵I]AII binding assay and insurmountable AT₁ receptor antagonism in the functional study.     

Are the serum levels of endotoxin-binding proteins reliable predictors of complications in the course of peritonitis?

Abstract. In the present study, the serum levels of the endotoxin-binding proteins transferrin, α₂-macroglo-bulin and Gc-globulin were nephelometrically determined in the morning of the first day after surgical treatment of peritonitis. The aim was to determine whether the occurrence of organ failure can be predicted by the serum levels of the described proteins. A serum level of transferrin below 1.15 g 1^-1 allows the prediction of organ failure with a sensitivity of 78.6% and a specificity of 81.8%. As in the case of α₂-macroglobulin, sensitivity and specificity reached 67.8% and 72.7% respectively at a border level of 1.05 g 1^-1. The predictive value of Gc-globulin, limit 0.2 g 1^-1, was in the same range. So the serum levels of these proteins allow an early prediction of forthcoming organ failure in the course of peritonitis. Furthermore, these results support the significance of the endotoxin interaction with these proteins.     

A 68-kD GTP-binding protein associated with the T cell receptor complex.

The identity of the guanine nucleotide-binding protein (G protein) involved in T cell activation pathways remains unclear. We identified a 68-kD GTP-binding protein associated with the T cell receptor (TCR)/CD3 complex using immunoprecipitation and GTP-affinity labeling techniques. Proteins coimmunoprecipitated with the TCR/CD3 complex in digitonin lysate of a human leukemic T cell line, MOLT 16, were incubated with alpha-[32P]GTP and irradiated with ultraviolet rays to covalently link the labeled GTP to GTP-binding proteins. They were then analyzed by electrophoresis. The 68-kD protein exhibited nucleotide specificity for GTP-binding and was insensitive to cholera and pertussis toxins. The 68-kD GTP-binding protein could be coimmunoprecipitated with the TCR/CD3 complex but not with other surface molecules such as major histocompatibility complex class I and lymphocyte function associated-1, which do not cause rapid Ca2+ mobilization. These suggest that the 68-kD GTP-binding protein is specifically associated with the TCR/CD3 complex.     

Correlation between renal membrane binding and nephrotoxicity of aminoglycosides.

The kinetics of aminoglycoside binding to renal brush border and basolateral membrane vesicles from rat renal cortex were studied by using [3H]amikacin. [3H]amikacin binding to renal membranes was found to be a rapid, saturable process with a fourfold greater affinity for basolateral membranes than for brush border membranes (Kd basolateral = 607 microM; Kd brush border = 2,535 microM). Renal membranes prepared from immature rats (2 to 3 weeks old) exhibited a significantly lower affinity compared with membranes from adults (Kd basolateral = 2,262 microM; Kd brush border = 6,216 microM). Additionally, the inhibitory behavior of several aminoglycosides versus [3H]amikacin binding to brush border membranes revealed the following rank order of potency: neomycin greater than tobramycin approximately gentamicin approximately netilmicin greater than amikacin approximately neamine greater than streptomycin. The relative insensitivity of immature rats to aminoglycoside-induced nephrotoxicity in vivo and the comparative nephrotoxicity of the various aminoglycosides suggest that renal membrane-binding affinity is closely correlated to the nephrotoxic potential of these antibiotics.     

Atrial Membranes Contain Nucleoside Diphosphate Kinase (NDPK) Activity: Its Role in Regulation of Muscarinic K+ Channels

Guinea pig or rabbit atrial muscarinic K⁺ channels in cell-free inside-out patches ran be activated in the absence of extracellular agonist and cytoplasmic G nucleotides by intracellular ATP-Mg²⁺,^1,2 This ATP-dependent activation is compatible with the existence of a membrane-bound nudeos de diphosphate kinase (NDPK), which directly phosphorylates G_K-bound GDP. We show that this ATP-dependent activation is also possible in frog atria] cells, and that atrail membranes of frog and guinea pig contain NDPK activity. The relative order of different nucleoside triphogphates (NTPs) as phosphate donors parallels the observed efficiency of these nucleotides in activation of the channels. Thus, atrial membranes contain NDPK activity, which can be responsible for the ATP-dependent activation o/muscarinic K⁺ channels, seen in patches of atrial cells. Under physiological conditions, NDPK can act as a GTP supply in the immediate vicinity of the G protein to ensure reliable signal transduction.